CD4+ T regulatory (Treg) that express the transcription factor FoxP3 cells play key and non-redundant roles in maintaining immunologic tolerance and controlling inflammation. Treg proportions within CD4+ T cells are under tight homeostatic control. There is considerable diversity to Treg cells, with differential ability to control certain immune functions, and even tissue-resident Tregs with extra-immunologic functions. Our recent recent single-cell RNAseq work has charted these finely, showing that Treg subsets express different effector molecules. Treg subset structure appears very similar in mouse and humans. We hypothesize that Treg subsets in lymphoid tissues respond to different homeostatic cues, and that these different inputs condition specific Treg functions. (1) Our single-cell transcriptomics data, which highlighted the diversity of Treg subphenotypes in an unbiased fashion, identified some cell surface markers for identification and sorting, but we will expand this base by performing a combined transcriptome/proteome analysis (scRNAseq combined with surface protein quantitation by DNA-tagged antibodies - CITE-seq) to identify a panel of flow cytometry markers that accurately distinguish Treg subphenotypes, in both mouse and human. This will relate this novel landscape to prior knowledge on Treg subsets, provide biomarkers, and enable cell sorting for functional experimentation. We will assess the function of Treg subsets in regulating other immunocytes (proliferation and differentiation), assess their developmental origin and stability (thymic or peripheral, plasticity?), and how they vary in autoimmune or tumor lesions. (2) IL2 is the canonical controller of Treg cells??but other common-? cytokines (IL7, 15, 21 and TSLP) and IL33 also affect Tregs. Our results show a strikingly divergent distribution of cytokine receptors among Treg subsets, suggesting that different growth factors support them. We will use both gain- (in vivo cytokines administration) or loss-of-function (blocking antibodies) strategies to modify Treg subset balance, read out by flow cytometry (STAT phosphorylation, Treg subset number and activation) and scRNAseq. (3) Most members of the large TNF Receptor (TNFR) superfamily are over-expressed in Treg cells, and some have been shown to affect Treg maintenance and survival, but a comprehensive picture is lacking. Our data show differential representation of TNFR across Treg subsets. We hypothesize that TNFR molecules network with cytokine receptors to provide specific trophic signals to distinct Treg subsets. We have developed a powerful CRISPR-based in vivo screen, based on barcoding stem cells, to evaluate how sets of genes partake in Treg homeostasis. We will analyze the role of the entire TNFR family on maintenance of different Treg subsets, at baseline in different lymphoid tissues and after stimulation by trophic cytokines. We will also test several TNFRs combinatorially, to search for redundancy, complementarity or antagonism. These results will provide a clarification and deeper understanding of the diversity of Treg populations, their control by cytokines or surface receptors, and how they might be specifically manipulated for therapeutic intent.